CN1294630A - Oligonucleotide sequences complementary to thioredoxin or thioredoxin reductase genes and methods of using same to modulate cell growth - Google Patents

Abstract

This invention relates to oligonucleotides complementary to the thioredoxin and thioredoxin reductase genes which modulate tumor cell growth in mammals. This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells in mammals. This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.

Description

Oligonucleotide sequence with Trx or thioredoxin reductase gene complementation

And the method for using it for the growth of adjusting cell

The relevant application of reference

The application requires the right of priority of the U.S. Provisional Application submitted on January 30th, 1998 number 60/073,196, and this sentences the reference mode and incorporates into this application is complete.

Background of the present invention

Field of the present invention

The present invention relates to and the Trx that can regulate the mammalian tumor cell growth and the oligonucleotide sequence of thioredoxin reductase gene complementation.The invention still further relates to the method that this compound is used to suppress the mammal tumor growth.The present invention relates to the pharmaceutical composition of the The compounds of this invention that contains pharmaceutical acceptable excipient and significant quantity again.

Reference

Quote following publication, patent application and patent with the subscript numeral:

1.Holmgren, " carrying out the enzyme process reduction-oxidation of protein disulfide by Trx " (Enzymology method) 107:295-300 (1984)

2.Wright and Anazodo, " antisense molecule and they are used for the treatment of the potentiality of cancer and acquired immune deficiency syndrome (AIDS) " cancer periodical 4:185-189 (1995)

3.Matthew etc., " the sulphur oxidation protein comprises the dna binding activity of the disulfide linkage adjusting NF-Kappa B of halfcystine 62 by reduction " nucleic acids research 20:3821-3830 (1992)

4.Bannister etc., " redox variable effect Fos/Jun and BZLF1 but do not influence the external dna binding activity of C/EBP " oncogene 6:1243-50 (1991)

5.Cromlish and Roeder " human transcription factor III C (TF III C) " biological chemistry periodical: 264:18100-9 (1989)

6.Abate etc., " redox modulating of the external dna binding activity of fos and Jus " science 249:1157-1161 (1990)

7.Tagaya etc., " ATL-derivative factor (ADF), a kind of and Trx homologous IL/2 acceptor/Tac inductor " European molecular biology periodical 8:757-64 (1989)

8.Powis etc., the control of cell growth " Trx/thioredoxin reductase reducto oxydative system unify " cancer research 6:539-44 (1994)

9.Oblong etc., " rite-directed mutagenesis of Trx's avtive spot halfcystine produces the competitive inhibitor of Trx's reductase enzyme and eliminates the short cell fission activity of Trx " biological chemistry periodical 269:11714-20 (1994)

10.Gasdaska etc., " redox protein matter Trx is by new help mechanism stimulate cell growth " cell Growth and Differentiation 6:1643-1650 (1995)

11.Gasdaska etc., " Trx's amino-acid sequence of prediction is identical with autocrine somatomedin adult T-cell derived factor (ADF); Trx mRNA increases in some people's tumour " biochemical and Acta Biophysica Sinica 1218:292-296 (1994)

12.Berggren etc., " expression in people's tumour and clone of Trx and thioredoxin reductase and the effect of serum stimulation and histanoxia " anticancer research 16:3459-3466 (1996)

13.Fujii etc., " Adult T-cell leukemia-derivative factor, a kind of Trx's homologue and the human papillomavirus HPV DNA coexpression in neoplastic neck tesselated epithelium " cancer 68:1583-91 (1991)

14.Kawahara etc., and " Trx and high movement property group albumen 1 gene in human hepatocellular carcinoma increase and with may getting in touch that the susceptibility of cis-platinum is reduced " cancer research 56:5330-3 (1996)

15.Gallegos etc., " increasing the conversion phenotype of the negative sudden change counter-rotating of the dominance human breast cancer cell of cell proliferation and Trx with Trx's transfection " cancer research 56:5765-70 (1996)

16.Baker etc., " Trx, the gene of discovery overexpression in people's cancer suppress external and intravital apoptosis " cancer research 57:5162-7 (1997)

17.Mau and Powis, " the inhibition of the cell thioredoxin reductase that diaziquone and Zorubicin cause.With the active relation of the ribonucleotide reductase of cell inhibitory effect and reduction " biochemical pharmacy 43:1621-7 (1992)

18.Mau and Powis, " antitumor quinonoid compound is to the inhibition based on mechanism of thioredoxin reductase " biochemical pharmacy 43:1613-20 (1992)

19.Schallreuter and Wood, " the melanomatous physiopathologic new aspect of skin: the function of sulfoprotein and to the summary of nitrosourea influence " melanoma research 1:159-167 (1991)

20.Schallreuter and Wod, " stereospecificity of human melanoma thioredoxin reductase by 13-cis-vitamin A acid committed suiside and suppressed " biochemical with biophysics research notes 160:573-9 (1989)

21.Curcio etc., " oligonucleotide is as the regulon of oncogene expression " pharmacological agent 74:317-32 (1997)

22.Narayanan and Akhtar, " antisense therapy " cancer present age comment 8:509-15 (1996)

23. the Lei Mingdun pharmaceutical science, Mace Publishing Company, Philadephia PA17 ^ThVersion (1985)

24.Sambrook etc., " molecular cloning: laboratory manual Cold Spring HarborLaboratory, New York (1989,1992)

25.Ausubel etc., molecular biology modernism John Wiley and Sons, Baltimore Maryland (1989)

26.Perbal, molecular cloning practical guide John Wiley ﹠amp; Sons, New York (1988)

27.Hurta and Wright, " the H-ras vicious transformation causes the unusual adjusting of the ribonucleotide reductase genetic expression that caused by transforming growth factor-beta " cellular biochemical periodical 57:543-556 (1995)

28.Tagaya etc., " ATL-derivative factor (ADF), a kind of and Trx homologous IL-2-acceptor/Tac inductor: may relate to the dimercapto reduction in the IL-2 receptor-inducible " European molecular biology periodical 13:2244 (1994)

29.Choy etc., " the drug resistance molecular mechanism that relates to ribonucleotide reductase: the hydroxyurea resistance in the relevant mouse cell line of a series of clones that under increasing the drug level condition, select " cancer research 48:2029-2038 (1988)

30.Fan etc., " ribonucleotide reductase R2 component is a kind of new pernicious determiner, and it and activated oncogene determine to transform and pernicious possibility jointly " periodical 93:14036-40 (1996) of institute of NAS

31.Huang and Wright, " drug resistance of fibroblast growth factor mediation changes and the gene amplification evidence " oncogene 9:491-499 (1994)

32.Gasdaska etc., " clone's of human thioredoxin albumen reductase enzyme order-checking " 373:5-9 (1995)

29.Nielsen etc.; Science (1991) 354:1497

30.Good and Nielsen; Periodical (1998) 95:2073-2076 of " peptide nucleic acid(PNA) of target ribosome-RNA(rRNA) causes the inhibition of translation and bacterial growth " institute of NAS

31.Buchardt, late, etc., U.S. Patent number No.5,766,855

32.Buchardt, late, etc., U.S. Patent number No.5,719,262

33. U.S. Patent number 5,034,506 ³³

34.Altschul, wait " basic part comparison research tool ", molecular biology magazine (1990) 215:403-10;

35.Devereux J, etc., " a series of comprehensive sequential analysis program that is used for VAX " nucleic acids research (1984) 12:387-395;

36.Chang etc. ³⁶Somatic gene therapy, CRC Press, Ann Arbor MI (1995);

37.Vega Deng 37; Gene targeting, CRC Press, Ann Arbor MI (1995)

38. carrier: molecular cloning vector and application thereof are scanned, Butterworths, Boston MA (1988)

39.Sullivan 1994, U.S. Patent number .5,225,347

40.1991 the United States Patent (USP) 5,023,252 that authorize on June 11,

41.Felgner etc., U.S. Patent number 5,580,859

42. United States Patent (USP) 5,011,472

43.Dreeley etc., science, 258:1650-1654 (1992)

Separately with the complete introducing of reference mode, incorporate all above-mentioned publications, patent application or patent into the present invention so that reference is complete as each publication, patent application or patent.

Prior art

Trx is a kind of little omnipresence redox protein matter, and confirming as at first is the reduced cofactor that DNA is synthesized very important ribonucleotide reductase ¹Trx and thioredoxin reductase constitute thioredoxin system.Thioredoxin reductase is a kind of flavo-enzyme that contains seleno-cysteine, and it uses NADPH to reduce Trx as protophobe, and Trx reduces other protein thereupon, and therefore influences its function.

In recent years, hinted that the Mammals Trx participates in multiple other biochemical route.For example, it regulates the redox property of transcription factor by dimercapto disulphide (dithiol disulfide) exchange, thereby changes their DNA binding characteristic.Transcription factor is such as NF-κ B ³, BZLFI ⁴With TF III C ⁵Directly regulated, and the activation of AP-1 is mediated indirectly by nuclear redox factor R ef-1, Ref-1 is further reduced by Trx ⁶In addition, shown that Trx can promote to contain the proteinic refolding of disulphide, activated glucocorticosteroid or interleukin-2-acceptor, HIV inhibiting is expressed in scavenger cell, reduces H ₂O ₂Remove free radical, protection cell antagonism oxidative pressure, and as a kind of early pregnancy factor.

The somatomedin that is called Adult T-cell leukemia's derivative factor that has shown clone's Trx and the T-cell release that HTLV-1 transforms is similar ⁷It uses a kind of approach secretion.The outer Trx of expressing of born of the same parents stimulates the propagation of normal fibroblast, lymphocyte and various human solid tumor cell system ^{8﹠amp; 9}Use redox not have form alive and show that growth-stimulating needs the redox active of Trx ⁹The growth-stimulating of Trx it seems by with cell to other somatomedin sensitization indirect induction ¹⁰Subsequently, report Trx overexpression in some primary tumo(u)r such as lung, colon, uterine neck and hepatocellular carcinoma ^11-14In addition, the human breast cancer cell with wild-type Trx cDNA transfection demonstrates the enhanced tumor growth ¹⁵, the spontaneous apoptosis of Jiang Diing in vivo ¹⁶And to multiple anticancer therapy compound inductive apoptosis, susceptibility reduces ¹⁶On the other hand, with the vTrxA AC cells transfected of dominance-feminine gender, redox-non-activity, in the external growth that does not rely on grappling that demonstrates reduction, and the tumor growth that demonstrates in vivo suppresses ¹⁵

The various human tumour demonstrates the overexpression of thioredoxin reductase ¹²Antineoplastic quinones ^17-18, the inhibition of nitrosourea and 13-cis-vitamin A acid pair cell thioredoxin reductase causes the active reduction of thioredoxin system, and causes the growth-inhibiting activity subsequently.

Use antisense oligonucleotide, suppressed genetic expression by sequence-specific hybridization with said target mrna with the target-specific form ²The oncogene of antisense oligonucleotide mediation suppresses to have disclosed the mechanism that these compounds can be used for identifying the domination oncogenesis, and also can promising new treatment compound as the treatment cancer ²²Thereby, needing to identify antisense oligonucleotide at Trx and thioredoxin reductase, it suppresses the expression of Trx and thioredoxin reductase with high specific and hypotoxicity.

The general introduction of invention

The present invention relates to regulate the antisense oligonucleotide of Trx and the expression of thioredoxin reductase gene in mammalian tumor cell, and the pharmaceutical composition that contains this antisense oligonucleotide.The invention still further relates to and use this class antisense oligonucleotide to suppress the method for growth of tumour cell and migration in Mammals.

Accordingly, aspect of its composition, the present invention relates to a kind of antisense oligonucleotide, this oligonucleotide contains about 3 to about 50 Nucleotide, this Nucleotide and mammiferous Trx mRNA or thioredoxin reductase mRNA complementation.Antisense oligonucleotide can be the nuclease resistance, and has the company's key between one or more thiophosphatephosphorothioate Nucleotide.Antisense oligonucleotide may further contain other not with Trx mRNA or thioredoxin reductase mRNA complementary Nucleotide.

Aspect another of composition, the present invention relates to a kind of containing from about 17 antisense oligonucleotides to about 50 Nucleotide, oligonucleotide wherein comprises the sequence 2601-2626[SEQ ID No:1-26 that selects shown in the Free Surface 1] sequence.

Aspect another of composition, the present invention relates to two kinds and contain from about 20 antisense oligonucleotides to about 50 Nucleotide, oligonucleotide wherein comprises the sequence 3001-3040[SEQ ID No:27-66 that selects shown in the Free Surface 2] sequence.

Aspect another of composition, the present invention relates to a kind of pharmaceutical composition, it contains the antisense oligonucleotide of pharmaceutical acceptable excipient and significant quantity, this oligonucleotide contains from about 3 to about 50 Nucleotide, this Nucleotide and mammiferous thioredoxin gene or thioredoxin reductase gene complementation.

Aspect another of composition, the present invention relates to a kind of pharmaceutical composition, it contains the antisense oligonucleotide of pharmaceutical acceptable excipient and significant quantity, this oligonucleotide contains from about 17 to about 50 Nucleotide, and oligonucleotide wherein comprises the sequence 2601-2626[SEQ ID No:1-26 that selects shown in the Free Surface 1] sequence.

Aspect another of composition, the present invention relates to a kind of pharmaceutical composition, it contains the antisense oligonucleotide of pharmaceutical acceptable excipient and significant quantity, this oligonucleotide contains from about 20 to about 50 Nucleotide, and oligonucleotide wherein comprises the sequence 3001-3040[SEQ ID No:27-66 that selects shown in the Free Surface 2] sequence.

Aspect of its method, the present invention relates to a kind of method that suppresses the mammal tumor growth, comprise the antisense oligonucleotide that under certain conditions suspection is had the administration significant quantity of tumour, make to suppress growth of tumor, this oligonucleotide comprise with about 3 Nucleotide of Mammals thioredoxin gene complementary to about 50 Nucleotide.Can be with antisense oligonucleotide with the chemotherapeutics administration.

Aspect another of method, the present invention relates to a kind of method that suppresses the mammal tumor growth, comprise the antisense oligonucleotide that suspection is had the administration significant quantity of tumour under certain condition, make to suppress growth of tumor, this oligonucleotide comprises that about 3 Nucleotide with Mammals thioredoxin reductase gene complementation are to about 50 Nucleotide.Can be with antisense oligonucleotide with the chemotherapeutics administration.

Aspect another of method, the present invention relates to a kind of method that suppresses the mammal tumor migration, comprise the antisense oligonucleotide that suspection is had the administration significant quantity of migration tumour under certain condition, make to suppress the migration of tumour, this oligonucleotide comprise with about 3 Nucleotide of Mammals thioredoxin gene complementary to about 50 Nucleotide.Can be with antisense oligonucleotide with the chemotherapeutics administration.

Aspect another of method, the present invention relates to a kind of method that suppresses the mammal tumor migration, comprise the antisense oligonucleotide that under certain conditions suspection is had the administration significant quantity of tumour, make tumor migration inhibition, this oligonucleotide comprise with about 3 Nucleotide of Mammals thioredoxin reductase gene complementation to about 50 Nucleotide.Can be with antisense oligonucleotide with the chemotherapeutics administration.

Brief description of the drawings

Figure 1A is the autoradiogram(ARGM) of Trx mRNA Northern trace in different clones: people's fetal tissues pneumonocyte system (WI-38), fibrosarcoma (HT-1080), lung cancer (A549), adenocarcinoma ovaries (SK-OV-3), hepatocellular carcinoma (HepG2), melanoma (C8161), mammary cancer (MDA-MB-231), transport property carcinoma of the pancreas (AsPC-1), adenocarcinoma of colon (HT-29), cervical cancer (HeLa S3).

Figure 1B is the photo of the proteinic Western trace of Trx of expressing in different clones.

Fig. 2 is the cDNA sequence [SEQ ID No:67 and 68] of Trx.26 kinds of different oligonucleotide annealed hybridization site have been marked.

The figure of Fig. 3 A-3F shows and uses with after 26 kinds of different antisense oligonucleotides of Trx cDNA complementary are handled that various human cancerous cell line colony forms the inhibition percentage ratio of ability.The clone that detects is human colon carcinoma HT-29 (Fig. 3 A), MCF-7 MDA-MB-231 (Fig. 3 B), human hepatoma cell line HepG2 (Fig. 3 C), human melanoma cell are A2058 (Fig. 3 D), and Proliferation of Human Ovarian Cell is SK-OV-3 (Fig. 3 E) and human lung cancer cell line A549 (Fig. 3 F).

Fig. 4 A is shown in to use with 6 kinds of antisense oligonucleotides of Trx mRNA complementary and handles the Trx mRNA level that reduces among the human hepatoma cell line HepG2s of back, represents with the percentage ratio of mRNA level in the relative untreated cell system.

Fig. 4 B is shown in to use with 6 kinds of antisense oligonucleotides of Trx mRNA complementary and handles the Trx mRNA level that reduces among the MCF-7 MDA-MB-231 of back, represents with the percentage ratio of mRNA level in the relative untreated cell system.

Fig. 5 A is after antisense oligonucleotide shown in demonstration is used is handled, the Western trace photo of Trx protein expression level in CCL188 HT-29.

Fig. 5 B is after antisense oligonucleotide shown in demonstration is used is handled, the Western trace photo of Trx protein expression level in MCF-7 MDA-MB-231.

Fig. 5 C shows the NO:1 with antisense oligonucleotide 2601[SEQ ID] handle after, the Western trace photo of Trx protein expression level in CCL188 HT-29, MCF-7 MDA-MB-231 and human hepatoma cell line HepG2.Below a hurdle show that the protein application of sample of each swimming lane is consistent.

Fig. 6 A is that the size of tumour in nude mice was to the mapping of time after every other day the antisense oligonucleotide intravenously was handled mouse shown in the usefulness.

Fig. 6 B be every other day with shown in the antisense oligonucleotide intravenously handled behind the mouse diagram of the weight of the tumour of removing from nude mice about 10 days.

Fig. 6 C diagram every other day with shown in the antisense oligonucleotide intravenously handled behind the mouse Western trace photo of Trx protein expression level from the human colon carcinoma HT-29 tumour of nude mice excision about 10 days.Show below and the protein application of sample is described with the painted part trace of Indian ink.

Fig. 7 is the autoradiogram(ARGM) of Northern trace, the thioredoxin reductase mRNA level of expressing in the tumor cell line shown in being presented at.

The figure of Fig. 8 A-8D shows and uses with after 40 kinds of different antisense oligonucleotides of thioredoxin reductase mDNA complementary are handled that various human cancerous cell line colony forms the inhibition percentage ratio of ability.The clone that detects is MCF-7 MDA-MB-231 (Fig. 8 A), human melanoma A2058 (Fig. 8 B), people's liver cancer HepG2 (Fig. 8 C) and human pancreas cancer SU.86.86 (Fig. 8 D).

Fig. 9 A and Fig. 9 B be shown in shown in antisense oligonucleotide handle thioredoxin reductase mRNA expression level in the clone of back, represent with the percentage ratio of mRNA level in the relative untreated cell system.Fig. 9 A is CCL188 HT-29, and Fig. 9 B is MCF-7 MDA-MB-231.

Figure 10 shows with antisense oligonucleotide 3014 and 3037[SEQ ID NO:40 and 63] handle after, the Western trace photo of thioredoxin reductase protein expression level in human pancreatic cancer cell AsPC-1.

Figure 11 A is that the size of people's tumour in nude mice was to the diagram of the mapping of time after every other day the antisense oligonucleotide intravenously was handled mouse shown in the usefulness.

Figure 11 B be every other day with shown in the antisense oligonucleotide intravenously handled behind the mouse diagram of the weight of people's tumour of removing from mouse about 10 days.

Figure 12 diagram every other day with shown in the antisense oligonucleotide intravenously handled behind the mouse Western trace photo of thioredoxin reductase protein expression level from the human colon carcinoma HT-29 tumour of nude mice excision about 10 days.

Detailed description definitions of the present invention

Following term used herein has following implication:

Term used herein " ASON " refers to the nuclear with required mRNA complementation Nucleotide sequence. Preferably, ASON and Trx mRNA or thioredoxin are also Protoenzyme mRNA complementation. The expection ASON can with 5 ＇ non-translational regions, the coding of mRNA Any complementation in the 3 ＇ non-translational regions of district or mRNA.

Term " oligonucleotides " refers to the nuclear that is made up of (skeleton) key between natural base, sugar and sugar The oligomer of thuja acid or nucleoside monomers or polymer. This term also comprises having containing of similar functions The modification of non-natural monomer or its part or replacement oligomer. Because the Cell uptake that for example strengthens or The features such as stability that tool increases in the presence of nuclease, relative day of this modification or replacement oligomer Right form is preferred. This term also comprises the chimeric few nuclear that contains two or more chemical distinct zones Thuja acid. For example, chimeric oligonucleotide can contain and gives useful feature (for example, the nuclease of increase Resistance is taken in the increase of cell) the zone of at least one modified nucleotide, maybe can be with this Bright two or more oligonucleotides connect the formation chimeric oligonucleotide.

ASON of the present invention can be ribonucleic acid or DNA, and can contain Natural existence or synthetic monomer base comprise adenine, guanine, cytimidine, thymidine And uracil. Oligonucleotides also can contain modified base, such as xanthine, and hypoxanthine, 2-amino Adenine, 6-methyl, 2-propyl group and other alkyl adenine, 5-halo uracil, born of the same parents are phonetic for the 5-halo Pyridine, 6-azauracil, 6-azepine cytimidine and 6-azathymine, pseudouracil, 4-sulfo-Uracil, 8-halo adenine, 8-aminoadenine, 8-sulfydryl adenine, 8-alkylthio group (thiolalkyl) adenine, the adenine that 8-hydroxyadenine and other 8-replace, 8-halo bird is fast Purine, the amino guanine of 8-, 8-thioguanine, 8-alkylthio group guanine, 8-hydroxyl guanine and its The guanine that its 8-replaces, other azepine and denitrogenation mix uracil, thymidine, cytimidine or bird Purine, 5-trifluoromethyl uracil and 5-three Flucytosines. Modify and also can comprise other chemical based Groups such as methyl, ethyl, propyl group of group appends to the different parts of oligonucleotides, comprise sugar, Base or skeleton component.

ASON of the present invention also can contain the phosphorus oxygen heteroatom of modification in the phosphoric acid skeleton, Key between key or short chain hetero atom or heterocycle sugar between short-chain alkyl or cycloalkyl sugar. For example, the few nuclear of antisense Thuja acid can contain methyl phosphonate, thiophosphate, phosphorodithioate, phosphotriester and Quinoline is for oligomer. In one embodiment of the invention, ASON contains and connects 4 to 6 The phosphorothioate bond of 3 ＇ terminal nucleotide. In another embodiment, phosphorothioate bond connects Connect all nucleotides. ASON also can have sugared analogies.

ASON of the present invention also can comprise nucleotide analog, wherein the structure of nucleotides Radical change is arranged. The example of this oligonucleotide analogs is peptide nucleic acid (PNA), wherein DNA Deoxyribose in (or RNA) (or ribose) phosphoric acid skeleton is by being similar to the polyamides of finding in the peptide The amine skeleton replaces. (Nielsen etc.²⁹ Good and Nielsen³⁰ Buchardt, late, etc.³¹ United States Patent (USP) 5,766,855; Buchardt, late, etc.³², United States Patent (USP) 5,719,262). PNA Analog has shown can resist enzyme degraded, and in vivo with the external life-span with prolongation. Because PNA chain and DNA interchain lack electrical charge rejection, and the combination of PNA and complementary dna sequence is also strong In with the combination of natural acid molecule.

Oligonucleotides of the present invention also can comprise that other contains polymer skeleton, cyclic skeleton or acyclic The nucleotides of skeleton. For example, nucleotides can contain morpholino skeleton structure (United States Patent (USP) 5,034,506³³)。

Thereby when being modified DNA and the RNA nuclease degradation is insensitive or they are put into protection When oligonucleotides is avoided the delivery vector of DNA and the effect of RNA nuclease, few nucleosides of the present invention Acid tool " nuclease resistance ": nuclease resistance oligonucleotides comprises, for example, and methyl phosphonate, Thiophosphate, phosphorodithioate, phosphotriester and morpholino oligomer. Give nuclease The suitable delivery vector of resistance comprises for example liposome.

Oligonucleotides of the present invention also can contain group, such as improving oligonucleotide drug dynamics spy The property group, or improve the group of oligonucleotides pharmacodynamic profiles. Preferably, oligonucleotides does not contain Reporter group or mark are arranged, such as fluorescent dye or radioactivity mark.

ASON preferably is selected from thioredoxin or thioredoxin reductase gene mutual The sequence of mending, double helix formation may appear in this sequence like this, hair clip forms and same oligomer/sequence That repeats may be minimum, but have high to moderate and thioredoxin or thioredoxin reductase The ability of gene order combination. These characteristics can be used computer modeling program OLIGO primer branch Analyse software (OLIGO Primer Analysis Software), version 5 (national Biological Science Co., Ltd (National Biosciences, Inc.) issue, Plymouth, MN). This computer program can To determine the qualitative estimation of these five kinds of parameters.

In addition, ASON also can be based on the sulphur oxygen between two or more mammalian species also The sequence of albumen or thioredoxin reductase gene is selecting of high conservative. These characteristics can Use computer group of University of Wisconsin (GCG) software (people such as Devereux J.³⁵) blast program (Aitschul waits the people³⁴) utilize state-run biotechnology information centre (NCBI) Database identification.

Antisense oligonucleotide can comprise sudden change, such as displacement, inserts and disappearance.Preferably will have and be lower than 10% sequence sudden change is arranged.

Antisense oligonucleotide generally contains about at least 3 Nucleotide or nucleotide analog, preferably from about 3 to about 100 Nucleotide or nucleotide analog, preferred from about 3 to about 50 Nucleotide or nucleotide analog, most preferred from about 17 to about 35 Nucleotide or nucleotide analog.

Preferably, antisense oligonucleotide contains the sequence of enumerating in table 1 and 2 (following).

Table 1

Has antisense oligonucleotide with Trx mRNA complementary sequence

Table 2 has the antisense oligonucleotide with Trx's reductase enzyme mRNA complementary sequence

In table 1 and table 2, " Tm " is the melting temperature(Tm) according to the oligonucleotide two strands of nearest neighbour thermokinetics numerical evaluation.50% nucleic acid molecule is double-stranded under this temperature, the 50%th, sex change." Δ G " is the free energy of oligonucleotide, is measuring of the double-stranded stability of oligonucleotide.

Term " alkyl " is meant the monovalent alkyl group, and 1 to 20 carbon atom is preferably arranged, and preferred 1 to 6 carbon atom.This term is illustrated as such as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, groups such as n-hexyl.

Term " aryl " is meant the unsaturated aromatic carbon ring group of 6 to 14 carbon atoms, contains monocycle (for example, phenyl) or a plurality of thick (fusion) ring (for example, naphthyl or anthryl).Preferred aryl groups comprises phenyl, naphthyl or the like.

Term " cycloalkyl " is meant the cyclic alkyl group of 3 to 20 carbon atoms, contains a monocycle or a plurality of condensed ring.This class group of naphthene base comprises, for example, single ring architecture is such as cyclopropyl, cyclobutyl, and cyclopentyl, ring octyl group or the like, or polynuclear plane is such as adamantyl or the like.

Term " halogen " or " halogen " are meant fluorine, chlorine, bromine and iodine, and fluorine or chlorine preferably.

Term " sulfydryl " is meant group-SH.

For containing one or more substituent any above-mentioned groups, should be appreciated that certainly this group does not contain three-dimensional unpractiaca and/or synthetic unsuitable any replacement or the substitute mode of.In addition, compound of the present invention comprises all because the three-dimensional chemical isomer that the replacement of these compounds produces.

Term " pharmacologically acceptable salts " is meant biological effect and the characteristic that keeps antisense oligonucleotide of the present invention, and and abiology or the unsuitable salt of others.Under many circumstances, antisense oligonucleotide of the present invention can owing to amino and/or carboxylic group or on it existence of similar group can form acidity and/or basic salt.

The acceptable base addition salt of pharmacy can be from inorganic and organic bases preparation.Comprise sodium from mineral alkali deutero-salt with way of example, potassium, lithium, ammonium, calcium and magnesium salts.Salt derived from organic bases include but not limited to primary amine, secondary amine and tertiary amine are such as alkylamine, dialkylamine, trialkylamine, substituted alkylamine, two (alkyl of replacement) amine, three (alkyl of replacement) amine, alkenyl amine, two alkenyl amines, three alkenyl amines, the alkenyl amine of replacement, two (alkenyl of replacement) amine, three (alkenyl of replacement) amine, Cycloalkyl amine, two (cycloalkyl) amine, three (cycloalkyl) amine, the Cycloalkyl amine of replacement, dibasic Cycloalkyl amine, trisubstituted Cycloalkyl amine, cycloalkenyl amine, two (cycloalkenyl) amine, three (cycloalkenyl) amine, the cycloalkenyl amine of replacement, dibasic cycloalkenyl amine, trisubstituted cycloalkenyl amine, arylamines, diarylamine, triarylamine, heteroaryl amine, two heteroaryl amine, three heteroaryl amine, heterocyclic amine, two heterocyclic amines, three heterocyclic amines, blended diamines and triamine, two substituting group differences on the amine at least wherein, and be selected from and comprise alkyl, the alkyl of replacement, alkenyl, the alkenyl that replaces, cycloalkyl, the cycloalkyl of replacement, cycloalkenyl, the cycloalkenyl that replaces, aryl, heteroaryl, heterocycle etc.Also comprise that two or three substituting groups form the amine of heterocycle or heteroaryl groups with amino nitrogen.

The example of suitable amine comprises, with way of example, and isopropylamine, Trimethylamine, diethylamide, three (sec.-propyl) amine, three (n-propyl) amine, thanomin, 2-dimethylaminoethanol, tromethamine, Methionin, arginine, Histidine, caffeine, PROCAINE HCL, PHARMA GRADE, hydrabamine, choline, trimethyl-glycine, 1, glycosamine, N-alkylated glucamine, Theobromine, purine, piperazine, piperidines, morpholine, N-ethylpiperidine or the like.Be to be understood that in practice of the present invention and can use other carboxylic acid derivative that for example, carboxylic acid amide comprises amides, low alkyl group acid amides, dialkyl amide or the like.

The acceptable acid salt of pharmacy can be from mineral acid and organic acid preparation.Salt derived from mineral acid comprise spirit of salt, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid, or the like.Salt derived from organic acid comprise acetate, propionic acid, oxyacetic acid, pyruvic acid, oxalic acid, oxysuccinic acid, propanedioic acid, succsinic acid, toxilic acid, fumaric acid, tartrate, citric acid, phenylformic acid, styracin, amygdalic acid, methylsulfonic acid, ethyl sulfonic acid, right-toluene sulfonic acide, Whitfield's ointment or the like.

Term " thioredoxin gene " is meant that coding can be used as proteinic any gene of the hydrogen donor of ribonucleotide reductase or methionine sulfoxide reductase.Preferred thioredoxin gene coding can be regulated the redox characteristic of transcription factor (such as NF-κ B, BZLF1, TF III C and AP-1), and changes the protein of their DNA binding characteristic.Other function that this protein can have includes but not limited to; promotion contains the ability of the proteinic refolding of disulfide linkage; activate the ability of glucocorticosteroid or interleukin-2 acceptor; or suppress the ability of the expression of immunodeficiency virus in scavenger cell, reduce water in the born of the same parents, remove free radical, the ability of protection cell antagonism oxidative pressure and as a kind of important component of early pregnancy factor.Preferably, thioredoxin gene a kind of protein of encoding, this protein can stimulate the propagation of normal fibroblast, lymphocyte and various human solid tumor cell system, and can stimulate tumor growth and reduce apoptosis during overexpression in people's tumour.

Term " thioredoxin reductase gene " is meant the proteinic any gene of NADPH-dependency reductive that coding can the catalysis Trx.

Term " with ... the complementation " meaning is that Antisensedigonucleotsequence sequence can be attached to target sequence, i.e. thioredoxin gene (or mRNA) or thioredoxin reductase gene (or mRNA).It is 75% identical that preferred Antisensedigonucleotsequence sequence and target sequence have at least, preferably at least 90% identical with target sequence, and most preferred at least 95% is identical, allows the breach of several bases or do not match.Homogeny for example can be determined with the BLASTN program of computer group of University of Wisconsin (GCG) software.As use OLIGO program described herein to confirm, preferred Antisensedigonucleotsequence sequence can with Trx or thioredoxin reductase mRNA at least 45 ℃ of melting temperature(Tm)s, more preferably at least at about 50 ℃ and most preferred at least 55 ℃ of hybridization.

Term " the suppress growth " meaning is that the growth of at least a tumor cell type reduces at least 10%, and is preferred at least 50%, and most preferred at least 75%.Reduction for growth of tumour cell can be determined by the size of measuring tumour in the nude mice, or can not determine at external formation colony by tumour cell.

Term " Mammals " or " mammiferous " are meant that all Mammalss comprise the people, sheep, ox, horse, pig, dog, cat and mouse etc.

One " suspection has the Mammals of tumour " meaning is that this Mammals has proliferative disease or tumour, or proliferative disease or tumour arranged after diagnosing, or proliferative disease or tumour had before been detected, and exenterate tumour, and suspect that this Mammals has some residual tumor cells.The preparation antisense oligonucleotide

Antisense oligonucleotide of the present invention can be by conventional and known technology preparation.For example, oligonucleotide can use the solid phase synthesis process preparation, particularly uses commercially available equipment such as Canadian Applied Biosystems, Inc., Mississauga, Canadian equipment preparation.Oligonucleotide also can prepare by naturally occurring Trx of means known in the art enzymatic degradation or thioredoxin reductase gene.The separation of antisense oligonucleotide and purifying

If desired, the separation of antisense oligonucleotide described herein can for example be filtered by any suitable separating and the purification process realization with purifying, extracting, crystallization, column chromatography, thin-layer chromatography, thick-layer chromatography, the combination of preparation type low pressure or high pressure liquid chromatography (HPLC) or these methods.But can certainly use other equivalent separation method.

With reference to the sequence of oligonucleotide and use means known in the art can make up the expression vector that contains Antisensedigonucleotsequence sequence.

Those skilled in the art can make up the carrier of the Expression element that contains all needs, transcribe to realize the required of Antisensedigonucleotsequence sequence.Like this, the invention provides the carrier of the transcriptional control sequence that contains the sequence that effectively is connected in the encoding antisense oligonucleotide.Suitable transcribe and translate element and can comprise bacterium from different sources, fungi, virus, Mammals or insect genes.Suitably selection of components depends on the host cell of selecting for use.

In carrier, can contain reporter gene.Suitable reporter gene comprises beta-galactosidase enzymes (being lacZ), E.C. 2.3.1.28, Lampyridea luciferase, or a kind of immunoglobulin (Ig) or its part.Can monitor transcribing of antisense oligonucleotide by the expression of monitoring reporter gene.

Can carrier be introduced cell or tissue by in the multiple currently known methods any.These class methods can be with reference to people such as Sambrook ²⁴Ausubel etc. ²⁵Chang ³⁶Vega etc. ³⁷And carrier: molecular cloning vector and application thereof ³⁸Summary etc., comprise for example stable or transient transfection, lipofection, electroporation and use recombining virus carrier infection.

Have many good qualities by infecting importing nucleic acid.Can efficiently obtain the specificity of types of organization.Virus generally infects in particular cell types and propagation.Like this, the specificity that can utilize virus with carrier in vivo target in the mixed culture of specific cell type or a kind of tissue or cell.Also available specific receptors or ligand modified virus vector are to change the targeting specific by receptor-mediated incident.

Oligonucleotide of the present invention can be insoluble.For example, oligonucleotide can be combined with appropriate carriers.The suitable carriers example is an agarose, Mierocrystalline cellulose, dextran, dextrane gel, sepharose, carboxymethyl cellulose polystyrene, filter paper, ion exchange resin, plastic film, plastics tubing, granulated glass sphere, polyamines-methyl ethylene-ether-maleic acid copolymerized body, the amino acid interpolymer, ethene-maleic acid copolymerized body, nylon, silk etc.Carrier can be for example manage, shape such as examination dish, pearl disk, sphere.

Soluble oligonucleotide can prepare for example cyanogen bromide coupling by using known chemistry or physical method that itself and suitable soluble carrier are reacted.

Expect that oligonucleotide of the present invention can be the ribozyme of a kind of mRNA of cutting.Ribozyme preferably contains and oligonucleotide sequence homologous sequence of the present invention and the essential catalytic center of cutting mRNA.For example, can select to destroy the homology ribozyme sequence of Trx or thioredoxin reductase mRNA.The ribozyme type that the present invention uses can be selected from type known in the art.Confirmed the structural family of multiple ribozyme, comprised I group intron, RNA enzyme P, hepatitis delta virus ribozymal, hammerhead ribozyme and from the hair clip ribozyme of nepovirus satellite RNA (sTRSV) (Sullivan 1994, United States Patent (USP) 5,225,347 ³⁹).Hammerhead ribozyme and the most normal transformation of hair clip ribozyme primitive are used for trans mRNA cutting, are used to carry out gene therapy (Sullivan 1994).The preferred hair clip ribozyme that uses among the present invention.Usually, ribozyme length is 30 to 100 Nucleotide.Pharmaceutical dosage form

During as medicine, use antisense oligonucleotide with the form of pharmaceutical composition usually.These compounds can pass through the different approaches administration, comprise oral, rectum, subcutaneous, intravenous through skin, intramuscular and nose in administration.These compounds are all effective as injection and oral compositions.These compositions prepare in the mode of knowing in the pharmacopedics technology, and contain a kind of active compound at least.Pharmaceutical composition for example can intravenous administration.Expection can be with pharmaceutical composition directly to the tumour administration that will treat.

The present invention also comprises this pharmaceutical composition, and it contains one or more antisense oligonucleotides as activeconstituents, and itself and pharmaceutical acceptable carrier or vehicle combine.In order to prepare composition of the present invention, usually with activeconstituents and mixed with excipients, with vehicle dilution or be embedded in the specific support, carrier can be the form of capsule, pouch, paper or other container.When vehicle during as thinner, it can be solid, semisolid or fluent material, as vehicle, carrier or the medium of activeconstituents.Like this, composition can be following form, tablet, pill, pulvis, lozenge, the bag agent, cachet, elixir, suspension agent, emulsion, solution, syrup, aerosol (solid or liquid medium) for example contains to the ointment of the active compound of 10% (weight), soft or hard ' Yanming ' capsules for clearing, suppository, aseptic parenteral solution, and the pulvis of sterile packed.

Preparation is during preparation, with may need active compound is ground to form suitable granular size before other composition mixes.If active compound is insoluble basically, be ground into usually less than 200 purpose granular sizes.If active compound is water soluble basically, regulates by grinding usually and can be provided at distribution unanimous on the whole in the preparation, for example about 40 orders.

The example of suitable vehicle comprises lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gel, Calucium Silicate powder, microcrystalline cellulose, polyvinylpyrrolidone, Mierocrystalline cellulose, sterilized water, syrup, methylcellulose gum.Preparation can comprise in addition: lubricant is such as talcum, Magnesium Stearate, and mineral oil; Wetting agent; Emulsification and suspension agent; Sanitas such as benzoic acid methyl ester and phenylformic acid hydroxy-propyl ester; Sweetner; And seasonings.Can be with composition of the present invention through preparation, to discharge at quick, the lasting or slowly-releasing that activeconstituents is provided after patient's administration by well known method.

Usually preferably composition is made the form of unitary dose, every dosage contains from about 3 milligrams of extremely about 3 grams, more frequent about 10 milligrams of extremely about 1.5 gram activeconstituentss.Term " unit dosage form " is meant the physically separated unit that is applicable to people experimenter and other Mammals unitary doses, per unit contains the active substance that can produce required result of treatment as calculated of the amount of pre-determining, and the drug excipient suitable with its bonded.

Antisense oligonucleotide is effective in very wide dosage range, and usually with the pharmacy effective dose administration.Significant quantity be can mitigation symptoms after the administration amount.Preferred significant quantity is to suppress the amount of growth of tumour cell.Preferred significant quantity is to about 20mg/kg body weight from about 0.1mg/kg body weight.Yet it must be understood that, the amount of the antisense oligonucleotide of actual administration will be by the doctor according to relevant environment, the amount, age, body weight, the reaction of individual patient, the severity of patient symptom that comprise the medication of the patient's condition to be processed, selection, actual administered compound, or the like determine.Can continue a couple of days the course of treatment to the several months, or alleviate up to obtaining disease.

In order to prepare solids composition such as tablet, main active ingredient/antisense oligonucleotide is mixed with drug excipient, the solid preparation that forms the uniform mixture that contains The compounds of this invention is determined preceding composition.Mention when these preparations determine that preceding composition is even, be meant that activeconstituents on average disperses in composition, thereby composition can be easy to be divided into equal effectively unit dosage form, such as tablet, pill and capsule.

Can be with tablet of the present invention or coating of pill or chemical combination so that the dosage form with prolongation effect advantage to be provided.For example, in tablet and pill can comprise and the external dose composition, the latter is by form with the former bag.Two kinds of compositions can be with the integument that can resist the degraded in the stomach separately, and complete components enters duodenum or postpones and discharges in making.Multiple material can be used as this class integument or dressing, and this class material comprises multiple polymeric acid and polymeric acid and such as lac, the mixture of cetyl alcohol and cellulose acetate etc.

Can mix new component of the present invention with in the liquid form that is applicable to oral or drug administration by injection, comprise the aqueous solution, suitable seasoning syrup, water or oil suspension, with the seasoning emulsion that uses edible oil (such as Semen Maydis oil, Oleum Gossypii semen, sesame oil, Oleum Cocois or peanut oil), and elixir and similar pharmaceutical carrier.

The composition that is used for sucking or be blown into comprises solution and at the suspension of acceptable water-based of pharmacy or organic solvent, or its mixture and pulvis.The liquid or solid composition can contain the acceptable vehicle of suitable pharmacy described herein.Preferably with composition through port or nasal respiration administration to produce part or systemic effect.Can use rare gas element with the composition aerosolization in the preferred medicine acceptable solvent.Can directly suck aerosol solution or the aerosol device is fitted on face shield or the intermittent positive pressure breathing respirator from the aerosol device.Solution, suspension or dust composition can be from preferred per os of the device of sending medicament in a suitable manner or nasal administrations.

The another kind of preferred formulation that uses in the inventive method uses transdermal delivery device (" patch ").This transdermal patch can be used for providing with controllable amount continuous or discontinuous the inculcating of antisense oligonucleotide of the present invention.Well knownly how to make up and use the transdermal patch that can be used for sending medicament.Referring to, for example, United States Patent (USP) 5,023,252 ⁴⁰, be incorporated herein by reference herein.This class patch can make up Rong in medicament continuously, pulsation or according to sending of requiring.

Another preferred delivering method relates to " air gun " that naked antisense oligonucleotide passes skin layer and sends.Sending of " naked " antisense oligonucleotide well known in the art.Referring to, for example, Felgner etc., United States Patent (USP) 5,580,859 ⁴¹Be expected at and antisense oligonucleotide can be packaged into lipid carrier before " air gun " sends antisense oligonucleotide.

Following example of formulations has been described typical pharmaceutical compositions of the present invention.

Example of formulations 1

Prepared the hard gel capsule that contains following ingredients:

Become component (mg/ capsule)

Activeconstituents 30.0

Starch 305.0

Magnesium Stearate 5.0

Mentioned component is mixed and be filled in the hard gel capsule with the amount of 340mg.

Example of formulations 2

Use following ingredients to prepare tablet:

Become component (mg/ sheet)

Activeconstituents 25.0

Mierocrystalline cellulose, crystallite 200.0

Silicon dioxide colloid 10.0

Stearic acid 5.0

The component mixing is compressed into tablet, each heavy 240mg.

Example of formulations 3

Prepare the Foradil Aerolizer formoterol fumarate preparation, contained following ingredients:

Composition weight %

Activeconstituents 5.0

Lactose 95.0

Activeconstituents and lactose are mixed, and mixture is added to powder inhaler.

Example of formulations 4

Tablet respectively contains the 30mg activeconstituents, following being prepared:

Become component (mg/ sheet)

Activeconstituents 30.0mg

Starch 45.0mg

Microcrystalline Cellulose 35.0mg

Polyvinylpyrrolidone 4.0mg

(in sterilized water, making 10% solution)

Sodium starch glycolate 4.5mg

Magnesium Stearate 0.5mg

Talcum 1.0mg

Amount to 120mg

Activeconstituents, starch and Mierocrystalline cellulose are passed No. 20 U.S. sieves (U.S.sieve) and mixing fully.Polyvinylpyrrolidonesolution solution is mixed with the pulvis of generation, pass U.S. sieve then No. 16.The particle that produces 50 ℃ to 60 ℃ dryings, and is passed U.S. sieve No. 16.Then will be in advance through the sodium starch glycolate of No. 30 U.S. sieves, Magnesium Stearate and talcum powder are added in the particle, mix the back produces each heavy 120mg on the tablet instrument tablet.

Example of formulations 5

Be prepared as follows the capsule that respectively contains the 40mg medicine:

Become component (mg/ capsule)

Activeconstituents 40.0mg

Starch 109.0mg

Magnesium Stearate 1.0mg

Amount to 150.0mg

With activeconstituents, starch and Magnesium Stearate mix, and pass U.S. sieve No. 20, are filled in the hard gel capsule with the amount of 150mg.

Example of formulations 6

Be prepared as follows the suppository that respectively contains the 25mg activeconstituents:

Become component

Activeconstituents 25mg

Saturated fatty acid glyceride to 2,000mg

Activeconstituents is passed U.S. sieve No. 60, be suspended in advance with minimum required adding in the saturated fatty acid glyceride that heat melts.The bolt mould that mixture is injected nominal 2.0g capacity cools off then.

Example of formulations 7

Be prepared as follows the suspension that every 5.0mL dosage contains the 50mg medicine:

Become component

Activeconstituents 50.0mg

Xanthan gum 4.0mg

Xylo-Mucine (11%)

Microcrystalline Cellulose (89%) 50.0mg

Sucrose 1.75g

Sodium Benzoate 10.0mg

Seasonings and pigment q.v.

Purified water is to 5.0mL

With activeconstituents, sucrose and xanthan gum are mixed, and pass U.S. sieve No. 10, mix with Microcrystalline Cellulose for preparing in water in advance and carboxymethylcellulose sodium solution then.The dilute with water Sodium Benzoate, seasonings and pigment also stir adding.Add enough water then and reach volume required.

Example of formulations 8

Become component (mg/ capsule)

Activeconstituents 15.0mg

Starch 407.0mg

Magnesium Stearate 3.0mg

Amount to 425.0mg

With activeconstituents, starch and Magnesium Stearate mix, and pass U.S. sieve No. 20, are filled in the hard gel capsule with the amount of 425.0mg.

Example of formulations 9

Can be prepared as follows preparation:

Become component

Activeconstituents 5.0mg

Semen Maydis oil 1.0mL

Example of formulations 10

Can be prepared as follows a kind of topical formulations:

Become component

Activeconstituents 1-10g

Emulsifying wax 30g

Whiteruss 20g

The white soft wax is to 100g

White soft wax is heated to thawing.Whiteruss and emulsifying wax are mixed and are stirred to dissolving.Add activeconstituents and continue to be stirred to dispersion.Then mixture is cooled to solid.

Other suitable preparation of available of the present invention can be referring to the Lei Mingdun pharmaceutical science ²³(Remington ' sPharmaceutical Sciences).

As long as essential material is wrapped into suitable containers, antisense oligonucleotide or the pharmaceutical composition that contains antisense oligonucleotide can be packaged into test kit easily.

Oligonucleotide of the present invention and the growth of ribozyme regulate tumor cell.Therefore the method for disturbing and suppress growth of tumour cell in the Mammals is provided, has comprised tumour or tumour cell are contacted with antisense oligonucleotide of the present invention.

Term " contact " is meant that with oligonucleotide ribozyme etc. are added to cell suspension or tissue samples, or with directly or indirectly cell or tissue administrations in animal such as oligonucleotide.

This method can be used for treating proliferative disease, comprises multi-form cancer, such as leukemia, and lymphoma (Hodgkins and non-Hodgkins), pernicious sarcoma, melanoma, adenoma, solid tissue cancer, the anoxic tumour, the squamous cell carcinoma of mouth, throat, larynx and lung, apparatus urogenitalis cancer such as cervical cancer and bladder cancer, hematopoiesis cancer, colorectal carcinoma, mammary cancer, pancreas cancer, kidney, the cancer of the brain, skin carcinoma, liver cancer, head and neck cancer and neural system cancer, and benign lesion is such as papilloma.Also comprise other proliferative disease, relate to the disease of arthrosclerosis, blood vessel hyperplasia and virus infection such as psoriasis and those.

Oligonucleotide of the present invention also can be used for treating drug-resistant tumors.The example of drug-resistant tumors is the tumour of anti-those chemotherapeutics (such as 5 FU 5 fluorouracil, ametycin, methotrexate or hydroxyurea) and high level expression is known can give its tumour to the P-glycoprotein of multiple cancer therapy drug (such as colchicine, vinealeucoblastine(VLB) and adriamycin) resistance; Or expression Dreeley etc. ⁴³The proteinic tumour of described multi-drug resistance.Accordingly, expection can be united oligonucleotide of the present invention and known anticancer compound or chemotherapeutics use or be added wherein.Chemotherapeutics is the compound that can suppress tumor growth.This class medicament includes, but are not limited to, 5 FU 5 fluorouracil, ametycin, methotrexate and hydroxyurea.The amount of expection chemotherapeutics is significant quantity (promptly being enough to suppress the amount of tumor growth) or is lower than significant quantity.

Find that oligonucleotide of the present invention can reduce the growth of transport property tumour (such as MDA-MB-231 mammary cancer, HT-29 adenocarcinoma of colon, A549 lung cancer and A2058 melanoma cancer cells).In one embodiment of the invention, provide and reduced the method that the transport property tumour is grown in Mammals, comprise and use a certain amount of and Trx mRNA or thioredoxin reductase mRNA complementary oligonucleotide, or use the oligonucleotide shown in table 1 and the table 2.

Can use virus or non-virus carrier oligonucleotide delivery.Sequence can be mixed in box or the construct, oligonucleotide of the present invention is expressed in cell.Preferably, construct contains suitable transcripting controling area oligonucleotide is recorded at transit cell.

Thereby carrier provided by the invention contains the transcriptional control sequence that effectively is connected with the sequence of code book invention oligonucleotide.The present invention further provides with these carrier transformed host cells, these host cells are selected from suitable eucaryon and prokaryotic cell prokaryocyte.

Known suitable carriers, and preferably it contains all essential Expression elements and transcribes to obtain the required of sequence.Phagemid is the specific example of the useful carrier of this class, because they can be used as plasmid or phage vector.The carrier example comprises, virus is such as phage, baculovirus, retrovirus, dna virus, liposome and other recombinant vectors.Carrier also can contain the element that is useful on protokaryon or eucaryon host system.Present technique field ordinary person knows which kind of host system is compatible with which kind of specific support.

Can carrier be introduced in the cell by stable or transient transfection, liposome transfection, electroporation and recombining virus carrier infection.

Can add that other characteristic guarantees its security and/or strengthens its curative effect to carrier.These characteristics comprise, for example, can be used for the negative mark of selecting with the cell of recombinant virus infection.The example of a this negative selectable marker is the TK gene, and it gives the susceptibility for antiviral 9-(1,3-dihydroxy-2-third oxygen methyl) guanine.Also can comprise and be limited in the characteristic of expressing in the particular cell types.This class feature comprises, for example, and promotor and the regulatory element single-minded to required cell type.

The reverse transcription carrier is the example that can be used for introducing in the body the another kind of carrier of required nucleic acid, because they provide such as advantages such as horizontal infection (lateral infection) and target specificitys.The cell that horizontal infection is single infection produces the process of a plurality of progeny virus particles that can infect flanking cell.The result infects bulk zone fast.

According to cell type, can select to be used for the carrier of the inventive method as target.For example, if need treatment mammary cancer, then can use epithelial cell specificity carrier.Similarly, if the cell of treatment hemopoietic system is preferred to the single-minded virus vector of hemocyte then.Use

Antisense oligonucleotide of the present invention can be used for multiple purpose.They can be used for suppressing the expression of thioredoxin gene in the mammalian cell, thereby suppress this cell growth.They can be used for suppressing thioredoxin reductase expression of gene in the mammalian cell, thereby suppress this cell growth.Oligonucleotide can be used as the existence that hybridization probe detects Trx mRNA in the mammalian cell or thioredoxin reductase mRNA.When used as such, but can be with oligonucleotide with suitable detection moiety (such as radio isotope, part, single-minded) mark in conjunction with another right member, biological example element.At last, oligonucleotide can be used as molecular weight marker.

In order to further specify the present invention and advantage thereof, provided certain embodiments below, but gone up in all senses and unrestricted claim.

Embodiment

In the following embodiments, all temperature are centigradetemperature (unless stated otherwise), and all percentage ratio is weight percentage (unless stated otherwise).

In the following embodiments, followingly write a Chinese character in simplified form following implication.If certain writes a Chinese character in simplified form undefined, it has its implication of accepting usually:

μ M=little rubbing

MM=milli rubs

M=mole

Ml=milliliter

μ l=microlitre

Mg=milligram

μ g=microgram

PAGE=polyacrylamide gel electrophoresis

Rpm=rotations per minute

Δ G=free energy, the measuring of oligonucleotide duplex stability

Kcal=kilocalorie

FBS=foetal calf serum

DTT=dithiothreitol (DTT)

SDS=sodium lauryl sulphate

PBS=phosphate buffered saline(PBS)

PMSF=phenylmethylsulfonyl fluoride molecular biology ordinary method:

The Protocols in Molecular Biology of standard well known in the art and do not specify usually according to Sambrook etc. ²⁴And Perbal ²⁶In description.Oligonucleotide

Antisense oligonucleotide is selected from and Trx mRNA or thioredoxin reductase mRNA complementary sequence, this sequence repeats minimum duplex formation, hair clip formation and homotype oligomer/sequence of may occurring like this, but with Trx mRNA sequence or thioredoxin reductase mRNA sequence high binding ability is arranged respectively.In addition, the mistake of having eliminated other sequence that often occurs or the tumor-necrosis factor glycoproteins in people and the mouse causes.These characteristics are to build program OLIGO by computer mould ^(R)The primer analysis software, 5.0 versions, (InternationalBiosciences, Inc.) Plymouth MN determines in international Biological Science Co., Ltd.About antisense oligonucleotide at Trx, selected 5 oligonucleotide (2601-2605) target due to 5 ＇ non-translational regions, 2 oligonucleotide (2606-2607) target is due near the translation starting point, 12 oligonucleotide (2608-2619) target is due to the coding region, and 7 oligonucleotide (2620-2626) and the hybridization of 3 ＇ non-translational regions.66 different antisense oligonucleotides have been designed altogether, then from (the Dalton Chemical Laboratories of dalton's chemical laboratory company, Inc.) (North York, Canada) or (the TriLink Biotechnologies of three biotech companies, Inc.) (San Diego CA.) orders.Clone

Obtaining people's fetal tissues pneumonocyte from U.S. typical case culture center (ATCC) is the WI-38 human cancer clone different with ten kinds, comprise fibrosarcoma (HT-1080), lung cancer (A549), adenocarcinoma ovaries (SK-OV-3), hepatocellular carcinoma (HepG2), melanoma (C8161), mammary cancer (MDA-MB-231), transport property carcinoma of the pancreas (AsPC-1), adenocarcinoma of colon (HT29), cervical cancer (HeLa S3), the human melanoma cell is A2058, human pancreas cancer SU.86.86.Clone be kept at α-MEM substratum of being supplemented with 10% foetal calf serum (FBS) (Gibco BRL, Gaithersburg, MD).The overexpression of embodiment 1 Trx in the human carcinoma cell line

To be added to tissue culturing plate from the equal portions cell suspension of different clones and grow to the Asia and be paved with (70-80%).Measure Trx mRNA or proteinic level by Northern and Western engram analysis.

(Hurta and Wright as previously mentioned ²³) carry out the Northern engram analysis after the minor modifications.At the appointed time use TRIzol reagent (Gibco BRL, Gaithersburg MD) from the total cell RNA of cell preparation.Classification RNA on 1.5% formaldehyde gel (10-20 μ g) also transfers to nylon membrane.With trace with ³²The 300bp PCR fragment hybridization of P-mark, this fragment is to use forward primer [SEQID NO:69] (5 ＇-CAG ATC GAG AGC AAG ACT G-3 ＇), reverse primer [SEQID NO:70] (5 ＇-TTC ATT AAT GGT GGC TTC AA-3 ＇) and with people liver 5 ＇-extension addition cDNA storehouse (Clontech, Palo Alto CA) is the template synthetic.The Trx nucleotide sequence information is from Genebank recording mechanism X77585.Trx mRNA is expressed as 520bp transcript (Tagaya etc. ²⁸) and (Molecular Dynamics, Sunnyvale CA) show with quantitative to use radioautograph or phosphorescence instrument (phosphorImager).

Detecting Glycerose triphosphoric acid desaturase (GADPH) mRNA simultaneously contrasts as the RNA application of sample.Carry out PCR once more, use forward primer [SEQ ID NO:71] (5 ＇-CGC GGG GCTCTC CAG AAC AT-3 ＇) and reverse primer [SEQ ID NO:72] (5 ＇-GCA ATGCCA GCC CCA GCG TC-3 ＇) to produce the dna probe of 308bp GADPH from above-mentioned identical cDNA storehouse.

Shown in Figure 1A was obvious, Trx mRNA had remarkable high-caliber expression than normal cell in all 9 kinds of different tumor cell lines.But the overexpression in various degree that in different clones, shows 1.5 to 5.6 times of scopes.

Whole-cell protein extract in preparation 2 times of sample pipetting volume damping fluids of 50-150 μ l (100mM Tris-HCl, pH6.8,0.2MDTT, 4%SDS, 20% glycerine and 0.015% tetrabromophenol sulfonphthalein).(Choy etc. as previously mentioned ²⁹With Fan etc. ³⁰) carry out the Western engram analysis after the minor modifications.Fractional separation protein extract on the 15%SDS-PAGE gel (10-20 μ g) is transferred to nitrocellulose filter and is shown with Indian ink dyeing.With anti-Trx antibody (0.2-1 μ g/ml) (American Diagnostica Inc., Greenwich is 1: 8 with dilution subsequently CT), the anti-goat IgG (Sigma of 000 horseradish peroxidase, St.Louis, MO) expression of detection Trx.(Amersham, Arlington Heights IL) demonstrate a kind of protein of about 12kDa by ECL.

Shown in Figure 1B,, between Trx mRNA and protein level, significant correlation is arranged because protein expression mode follows observed intensity of variation in the mRNA expression pattern closely.The following hurdle of Figure 1B is presented at that the gross protein application of sample is consistent in the swimming lane.Embodiment 2. and the growth of Trx complementary antisense oligonucleotide anticancer system

Use foregoing method (Choy etc. ²⁹) colony of having estimated the cancerous cell line of handling with 26 kinds of different antisense oligonucleotides forms ability.Specific, with the tumour cell suspension of equal portions with roughly 1 * 10 ⁴Density be inoculated into tissue culturing plate, and in being supplemented with α-MEM of 10%FBS 37 ℃ of overnight incubation.With 5ml PBS flushing cell once, and cation lipid (lipofection reagent, final concentration 5 μ g/ml, Gibco-BRL, Gaithersburg MD) exists down with the appointment antisense oligonucleotide processing of 0.2 μ M 4 hours.Once remove antisense oligonucleotide with PBS flushing cell, cell (was supplemented with among the α of 10%FBS-MEM) cultivation 7 to 10 days at 37 ℃ of following growth mediums.With methylene blue colony is dyeed, and as (Choy etc. ²⁹And Huang and Wright ³¹) described direct census evaluation.By with do not contain the antisense oligonucleotide condition under the colony number that exists in the culture compare, calculate and suppress percentage ratio.All experiments are four parts of repetitions.

Antisense oligonucleotide demonstrates retarding effect to human tumor cell line colony formation ability.The inhibition percentage ratio of each antisense oligonucleotide is shown among Fig. 3, and Fig. 3 A is to CCL188 HT-29; Fig. 3 B is to MCF-7 MDA-MB-231; Fig. 3 C is to the human hepatoma cell line HepG2; Fig. 3 D is to be A2058 to the human melanoma cell; Fig. 3 E is to be SK-OV-3 to Proliferation of Human Ovarian Cell; Fig. 3 F is to human lung cancer cell line A549.Embodiment 3 uses with Trx complementary antisense oligonucleotide and handles the mRNA level that the back reduces

Human liver cancer cell (HepG2) or human breast cancer cell (MDA-MB-231) are cultured to the Asia are paved with (70-80%), and at cation lipid (lipofection reagent, final concentration 5 μ g/ml Gibco-BRL) and under Opti-MEM (Gibco-BRL) existence handled 4 hours with 0.2 μ M and Trx complementary phosphorothioate antisense oligonucleotide.With PBS flushing cell once, and with cell in being supplemented with the α of 10%FBS-MEM substratum, cultivated 16 hours.The total RNA of preparation also carries out Northern trace mensuration as previously mentioned in TRIzol reagent (Gibco-BRL).Trx mRNA level is quantitative, and to GAPDH mRNA horizontal alignment, be expressed as from the percentage ratio of the Trx mRNA level of untreated cell acquisition.Fig. 4 A and 4B show that antisense oligonucleotide reduces Trx mRNA level to being at least 50% of control cells.Embodiment 4 uses the reduction of handling back Trx protein expression in different clones with Trx complementary antisense oligonucleotide

Different clones are cultured to the Asia are paved with (70-80%), and (lipofection reagent, final concentration 5 μ g/ml Gibco-BRL) and under Opti-MEM (Gibco-BRL) existence handled 4 hours with 0.2 μ M phosphorothioate antisense oligonucleotide at cation lipid.Once and with cell in containing the α of 10%FBS-MEM substratum (Gibco-BRL), cultivated 20 hours with PBS flushing cell.Re-treatment and cultivate once after, full cell extract of preparation and (Choy etc. as previously mentioned in 2X sample loading buffer (100mM Tris-HCl, pH6.8,0.2M DTT, 4%SDS, 20% glycerine and 0.015% tetrabromophenol sulfonphthalein) ²⁹With Fan etc. ³⁰) carry out the Western trace after the minor modifications and detect.With anti-Trx antibody (0.2-1 μ g/ml) (American Diagnostica Inc., Greenwich is 1: 8 with dilution subsequently CT), the anti-goat IgG (Sigma of 000 horseradish peroxidase, St.Louis, MO) expression of detection Trx.(Amersham, Arlington Heights IL) demonstrate a kind of protein of about 12kDa by ECL.

The clone that detects is human colon cancer cell HT-29 (Fig. 5 A) and MDA-MB-231 human breast cancer cell (Fig. 5 B).With shown in antisense oligonucleotide reduced protein level after handling.

Use 0.2 μ M oligonucleotide 2601[SEQ ID NO:1 as mentioned above] handled three kinds of different human tumor cells (being respectively colon, the breast and the HT-29 of liver cancer, MDA-MB-231 and HepG2 clone).Fig. 5 C is presented at antisense oligonucleotide inhibition Trx protein expression in each clone.A lower hurdle shows that the protein application of sample is consistent in swimming lane.Embodiment 5 will with Trx complementary antisense oligonucleotide intravenous injection mouse after to the inhibition of growth of human tumor cells

Buy the CD-1 nude mouse from Charles River Laboratories (Montreal Canada).The HT-29 human colon cancer cell (is typically had 3 * 10 in 100 μ l PBS ⁶Cell) is subcutaneously injected into the right side of the female nude mice of 6-7 week CD-1 athymia in age.Each experimental group comprises 5 mouse.When the size of tumour reaches about 100mm ³Volume the time, typically after 5 days, different antisense oligonucleotides every other day is filled into the tail intravenously administrable with the amount heavy dose of 10mg/kg at tumor cell injection.The contrast mouse is only accepted salt solution in the same period.After this treatment generally continues 10 days.

Fig. 6 A shows the effect of antisense oligonucleotide to HT-29 tumor growth in the CD-1 nude mice.Estimate anti-tumor activity by suppressing gross tumor volume, this is to use calipers average two days mensuration at interval in 9 day time to measure.Each point among the figure is meant the mean tumour volume that calculates from 5 mouse of each experimental group.Use covariance analysis with the regression curve of mouse in each treatment group relatively to the time.Drawing slope when slope is identical from analyze equates or the identical concrete supposition of intercept.SAS (statistical analysis system) version 6.12 is used in all analyses.Fig. 6 A shows that each antisense oligonucleotide of comparing with saline control suppresses growth of tumor, p value≤0.0001.

Treatment finishes back (last usually treatment is after 24 hours) kill animals, measures tumor weight.Fig. 6 B shows the weight in average of tumour.Antisense oligonucleotide demonstrates the remarkable retarding effect to tumor growth.Used the mean value of the monolateral analysis comparison process group of variance.When the effect of all groups was remarkable, it was right to use the priori multiple comparisons (priori multiple comparison) that has utilized minimum Fang Jun (least square means) to seek the treatment group that there were significant differences.During comparison of tumor weight, compare each antisense oligonucleotide with saline control and also show inhibition significantly on the statistics, p value≤0.0198 (Fig. 6 B).

The HT-29 human colon cancer cell is subcutaneously injected into the right side of the female nude mice of 6-7 week CD-1 athymia in age.When the size of tumour reaches about 100mm ³Volume the time, generally after 5 days, different antisense oligonucleotides every other day is filled into the tail intravenously administrable with the amount heavy dose of 10mg/kg at tumor cell injection.The contrast mouse is only accepted salt solution in the same period.Kill mouse after 8 injections and collect the similar size tumor piece that downcuts rapidly, put into RIPA extraction buffer (50mM Tris-HCl, pH7.5,150mM NaCl, 1%NP-40,0.5% Sodium desoxycholate, 0.1%SDS, 0.02%NaN3,1mM PMSF and 10 μ M leupeptins) also homogenate is used for the protein preparation fast.In order to measure the effect of antisense oligonucleotide to the Trx protein level, as previously mentioned (Choy etc. ²⁹With Fan etc. ³⁰) carry out the Western engram analysis after the minor modifications.Fractional separation protein extract on the 12%SDS-PAGE gel (10-20 μ g) is transferred to nitrocellulose filter and is shown with Indian ink dyeing.With anti-Trx antibody (0.2-1 μ g/ml) (AmericanDiagnostica Inc., Greenwich is 1: 8 with dilution subsequently CT), the anti-goat IgG (Sigma of 000 horseradish peroxidase, St.Louis, MO) expression of detection Trx.(Amersham, Arlington Heights IL) demonstrate a kind of protein of about 50kDa by ECL.The protein of each swimming lane application of sample is roughly the same.Shown with the painted part trace of Indian ink identical application of sample is arranged below to confirm each swimming lane.Can see clearly from Fig. 6 C, compare that being expressed in the tumor tissues that obtains of Trx significantly reduces from the mouse that the antisense oligonucleotide that schedules Trx with target is handled with the control tumor tissue that obtains from the brine treatment mouse.Embodiment 6 sulphur oxygen are the egg reductase enzyme overexpression in the human carcinoma cell line in vain also

To be added to tissue culturing plate from the equal portions cell suspension of different clones and grow to the Asia and be paved with (70-80%).Measure thioredoxin reductase mRNA level by the Northern engram analysis.

(Hurta and Wright as previously mentioned ²³) carry out the Northern engram analysis after the minor modifications.In brief, at the appointed time use TRIzol reagent (Gibco BRL, Gaithersburg MD) from the total cell RNA of cell preparation.Fractional separation RNA on 1.5% formaldehyde gel (10-20 μ g) also transfers to nylon membrane.With trace with ³²The 330bp PCR fragment hybridization of P-mark, this fragment is to use forward primer [SEQ ID NO:73] (5 ＇-TTG GCT TAG AAA CCG TAG GG-3 ＇), reverse primer [SEQ ID NO:74] (5 ＇-CCA ATG GCC AAA AGT AACTA-3 ＇) and people liver 5 ＇-extension addition cDNA storehouse (Clontech, Palo Alto CA) is the template synthetic.Trx's reductase enzyme mRNA is expressed as the transcript (Gasdaska etc. of 3826bp ³²) and (Molecular Dynamics, Sunnyvale CA) manifest with quantitative to use radioautograph or phosphorescence instrument.

Detecting Glycerose triphosphoric acid desaturase (GADPH) mRNA simultaneously contrasts as the RNA application of sample.Carry out PCR once more, use forward primer [SEQ ID NO:71] (5 ＇-CGC GGG GCTCTC CAG AAC AT-3 ＇) and reverse primer [SEQ ID NO:72] (5 ＇-GCA ATGCCA GCC CCA GCG TC-3 ＇) to produce the dna probe of 308bpGADPH from above-mentioned same cDNA storehouse.

As Fig. 7 obviously shown in, thioredoxin reductase mRNA in all 9 different tumor cell lines than in the normal cell system remarkable high-caliber expression being arranged.Embodiment 7. and the growth of thioredoxin reductase complementary antisense oligonucleotide anticancer system

Use foregoing method (Choy etc. ²⁹) colony of having estimated the cancerous cell line of handling with 40 kinds of different antisense oligonucleotides forms ability.Specific, with the tumour cell suspension of equal portions with roughly 1 * 10 ⁴Density be inoculated into tissue culturing plate, and in being supplemented with α-MEM of 10%FBS 37 ℃ of overnight incubation.With 5ml PBS flushing cell once, and cation lipid (lipofection reagent, final concentration 5 μ g/ml, Gibco-BRL, Gaithersburg MD) exists down with the appointment antisense oligonucleotide processing of 0.2 μ M 4 hours.Once remove antisense oligonucleotide with PBS flushing cell, and cell (was supplemented with among the α of 10%FBS-MEM) cultivation 7 to 10 days at 37 ℃ of following growth mediums.With methylene blue colony is dyeed and as (Choy etc. ²⁹And Huang and Wright ³¹) described direct census evaluation.By with do not contain the antisense oligonucleotide condition under the colony number that exists in the culture compare to calculate and suppress percentage ratio.All experiments are four parts of repetitions.

Most of antisense oligonucleotide shows the retarding effect of moderate to human tumor cell line colony formation ability.The inhibition percentage ratio of each antisense oligonucleotide is shown among Fig. 8, and Fig. 8 A is to MCF-7 MDA-MB-231; Fig. 8 B is to be A2058 to the human melanoma cell; Fig. 8 C is to the human hepatoma cell line HepG2; Fig. 8 D is to be SU.86.86 to people's pancreatic cancer.Embodiment 8 uses with thioredoxin reductase complementary antisense oligonucleotide and handles the mRNA level that the back reduces

Human colon cancer cell (HT-29) or breast cancer cell (MDA-MB-231) are cultured to the Asia are paved with (70-80%), and at cation lipid (lipofection reagent, final concentration 5 μ g/ml Gibco-BRL) and under Opti-MEM (Gibco-BRL) existence handled 4 hours with 0.2 μ M and Trx complementary phosphorothioate antisense oligonucleotide.Once and with cell in being supplemented with the α of 10%FBS-MEM substratum, cultivated 16 hours with PBS flushing cell.The total RNA of preparation in TRIzol reagent (Gibco-BRL), and carry out the Northern trace as previously mentioned and measure.Trx's reductase enzyme mRNA level is quantitative, and to GAPDH mRNA horizontal alignment, be expressed as from the percentage ratio of the thioredoxin reductase mRNA level of untreated cell acquisition.Fig. 9 A and 9B show that antisense oligonucleotide reduces thioredoxin reductase mRNA level to being at least 50% of control cells.Embodiment 9 uses antisense oligonucleotide to handle back also reduction of albumen reductase enzyme protein matter expression of sulphur hydrogen in different clones

AsPC-1 people's pancreatic cancer is cultured to the Asia is paved with (70-80%), and at cation lipid (lipofection reagent, final concentration 5 μ g/ml, Gibco-BRL) and Opti-MEM (Gibco-BRL) exist down with 0.2 μ M phosphorothioate antisense oligonucleotide 3014[SEQ ID NO:40] and 3037[SEQ ID NO:63] processing 4 hours.With PBS flushing cell once, and with cell in containing the α of 10%FBS-MEM substratum (Gibco-BRL), cultivated 20 hours.Re-treatment and cultivate once after, preparation whole-cell protein matter extract in 2X sample loading buffer (100mM Tris-HCl, pH6.8,0.2M DTT, 4%SDS, 20% glycerine and 0.015% tetrabromophenol sulfonphthalein), and (Choy etc. as previously mentioned ²⁹With Fan etc. ³⁰) carry out the Western trace after the minor modifications and detect.With anti-thioredoxin reductase antibody (0.2-1 μ g/ml) (Research Genetics, Inc., Huntsville AL) it is 1: 8 with dilution subsequently, anti-goat IgG (the Sigma of 000 horseradish peroxidase, St.Louis, MO) expression of detection thioredoxin reductase.(Amersham, Arlington Heights IL) demonstrate a kind of protein of about 50kDa, referring to Figure 10 by ECL.Embodiment 10 usefulness antisense oligonucleotides are handled the inhibition of back to growth of tumour cell in the mouse

The HT-29 human colon cancer cell (is generally had 3 * 10 in 100 μ l PBS ⁶Cell) is subcutaneously injected into the right side of the female nude mice of 6-7 week CD-1 athymia in age.When the size of tumour reaches about 100mm ³Volume the time, generally after 5 days, different antisense oligonucleotides every other day is filled into the tail intravenously administrable with the amount heavy dose of 10mg/kg at tumor cell injection.The contrast mouse is only accepted salt solution in the same period.After this treatment typically continues 10 days.

Figure 11 A shows and the effect of thioredoxin reductase complementary antisense oligonucleotide to HT-29 tumor growth in the CD-1 nude mice.Estimate anti-tumor activity by suppressing gross tumor volume, this be to use calipers in 9 day time with two days at interval average measurement measure.The mean tumour volume that each some representative among the figure is calculated from 5 mouse of each experimental group.Treatment finishes back (last usually treatment is after 24 hours) kill animals, measures tumor weight.Figure 11 B shows the weight in average of tumour.Antisense oligonucleotide demonstrates the remarkable retarding effect to tumor growth.When comparing with saline control, each antisense oligonucleotide shown in Figure 11 A suppresses growth of tumor, p value≤0.0001.During comparison of tumor weight, compare each antisense oligonucleotide with saline control and also show inhibition significantly on the statistics, p value≤0.0141 (Figure 11 B).Embodiment 11 is with the reduction of thioredoxin reductase protein level in the antisense oligonucleotide intravenous injection mouse descendant tumour

The HT-29 human colon cancer cell (is generally had 3 * 10 in 100 μ l PBS ⁶Cell) is subcutaneously injected into the right side of the female nude mice of 6-7 week CD-1 athymia in age.When the size of tumour reaches about 100mm ³Volume the time, generally after 5 days, different antisense oligonucleotides every other day is filled into the tail intravenously administrable with the amount heavy dose of 10mg/kg at tumor cell injection.The contrast mouse is only accepted salt solution in the same period.Kill mouse after 8 injections, and collect the similar size tumor piece that downcuts rapidly and put into RIPA extraction buffer (50mM Tris-HCl, pH7.5,150mM NaCl, 1%NP-40,0.5% Sodium desoxycholate, 0.1%SDS, 0.02%NaN ₃, 1mM PMSF and 10 μ M leupeptins), homogenate is used for the protein preparation fast.In order to measure the effect of antisense oligonucleotide to the thioredoxin reductase protein level, (Choy etc. as previously mentioned ²⁹With Fan etc. ³⁰) carry out the Western engram analysis after the minor modifications.Fractional separation protein extract on the 12%SDS-PAGE gel (10-20 μ g) is transferred to nitrocellulose filter and is shown with Indian ink dyeing.With anti-thioredoxin reductase antibody (0.2-1 μ g/ml) (Research Genetics, Inc., Huntsville AL) it is 1: 8 with dilution subsequently, anti-goat IgG (the Sigma of 000 horseradish peroxidase, St.Louis, MO) expression of detection thioredoxin reductase.(Amersham, Arlington Heights IL) demonstrate a kind of protein of about 50kDa by ECL.The protein of each swimming lane application of sample is roughly the same.Referring to Figure 12.

Claims (26)

1. one kind contains from about 17 antisense oligonucleotides to about 50 Nucleotide, and oligonucleotide wherein comprises the sequence 2601-2626[SEQ ID NO:1-26 that is selected from shown in the table 1] sequence.

2. the antisense oligonucleotide of claim 1, it also comprises key between one or more thiophosphatephosphorothioate Nucleotide.

3. the antisense oligonucleotide of claim 1, its also comprise other not with Trx mRNA complementary Nucleotide.

4. one kind contains from about 17 antisense oligonucleotides to about 50 Nucleotide, and oligonucleotide wherein comprises the sequence 3001-3040[SEQ ID NO:27-66 that is selected from shown in the table 2] sequence.

5. the antisense oligonucleotide of claim 4, it also comprises key between one or more thiophosphatephosphorothioate Nucleotide.

6. the antisense oligonucleotide of claim 4, its also comprise other not with thioredoxin reductase mRNA complementary Nucleotide.

7. a carrier contains and comprises from about 3 oligonucleotide sequences to about 50 Nucleotide this Nucleotide and mammiferous Trx mRNA or thioredoxin reductase mRNA complementation.

8. pharmaceutical composition, it contains the antisense oligonucleotide of acceptable vehicle of pharmacy and significant quantity, this antisense oligonucleotide comprises the oligonucleotide sequence that contains from about 3 to 50 Nucleotide, this Nucleotide and mammiferous Trx mRNA or thioredoxin reductase mRNA complementation.

9. the pharmaceutical composition of claim 8, wherein antisense oligonucleotide contains from about 17 to about 50 Nucleotide, and contain be selected from sequence 2601-2626[SEQ ID NO:1-26 as shown in table 1] sequence.

10. the pharmaceutical composition of claim 8, wherein antisense oligonucleotide contains from about 17 to about 50 Nucleotide, and contain be selected from sequence 3001-3040[SEQ ID NO:27-66 as shown in table 2] sequence.

11. method that suppresses the mammal tumor growth, comprise the antisense oligonucleotide that the administration significant quantity of tumour is arranged to suspection under certain conditions, make tumor growth be suppressed, antisense oligonucleotide contain with Mammals Trx mRNA complementary about 3 to about 50 Nucleotide.

12. the method for claim 11, it also comprises the step to the administration chemotherapeutics.

13. the method for claim 11, oligonucleotide wherein contain the sequence 2601-2626[SEQ ID NO:1-26 that is selected from shown in the table 1] sequence.

14. the method for claim 11, oligonucleotide wherein are the nuclease resistances.

15. method that suppresses the mammal tumor growth, comprise the antisense oligonucleotide that the administration significant quantity of tumour is arranged to suspection under certain conditions, make tumor growth be suppressed, antisense oligonucleotide contain with about at least 3 Nucleotide of Mammals thioredoxin reductase gene complementation to about 50 Nucleotide.

16. the method for claim 15, it also comprises the step to the administration chemotherapeutics.

17. the method for claim 15, oligonucleotide wherein are the nuclease resistances.

18. the method for claim 15, oligonucleotide wherein contain the sequence 3001-3040[SEQ ID NO:27-66 that is selected from shown in the table 2] sequence.

19. method that suppresses the mammal tumor migration, comprise the antisense oligonucleotide that the administration significant quantity of transport property tumour is arranged to suspection under certain conditions, make the migration of tumour be suppressed, antisense oligonucleotide contain with about 3 Nucleotide of Mammals thioredoxin gene complementary to about 50 Nucleotide.

20. the method for claim 19, it also comprises the step to the administration chemotherapeutics.

21. method that suppresses the mammal tumor migration, comprise the antisense oligonucleotide that the administration significant quantity of transport property tumour is arranged to suspection under certain conditions, make the migration of tumour be suppressed, antisense oligonucleotide contain with about 3 Nucleotide of Mammals thioredoxin reductase gene complementation to about 50 Nucleotide.

22. the method for claim 21, it also comprises the step to the administration chemotherapeutics.

23. an oligonucleotide is used to suppress the purposes of growth of tumour cell, this oligonucleotide contain with mammiferous Trx mRNA or thioredoxin reductase mRNA complementary about 3 to about 50 Nucleotide.

24. an oligonucleotide is used to the purposes that suppresses to move, this oligonucleotide contain with mammiferous Trx mRNA or thioredoxin reductase mRNA complementary about 3 to about 50 Nucleotide.

25. an oligonucleotide is used to prepare the purposes of the medicine that suppresses growth of tumour cell, this oligonucleotide contain with mammiferous Trx mRNA or thioredoxin reductase mRNA complementary about 3 to about 50 Nucleotide.

26. an oligonucleotide is used to prepare the purposes of the medicine that suppresses migration, this oligonucleotide contain with mammiferous Trx mRNA or thioredoxin reductase mRNA complementary about 3 to about 50 Nucleotide.

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